Nicotine, its metabolites and related tobacco alkaloids are implicated as causative agents in diseases which are statistically more prevalent among smokers than non-smokers, e.g., lung cancer, cardiovascular disorders, decreased fetal size and increased fetal complications. The metabolism of nicotine and related compounds is complex and many products are formed. In order to better understand the effects of parent compounds and metabolites, it is important to have sensitive and specific assays to monitor levels of these compounds in physiological fluids and tissue extracts. Such assays are also indispensable in experiments (enzymatic and chemical) designed to determine the factors which influence the formation and catabolism of individual compounds. Specific and sensitive radioimmunoassays are available for nicotine, cotinine, gamma (3-pyridyl)-gamma-oxo-N-methylbutyramide, N-nitrosonornicotine and the NMN, NAD and NADP analogs of nicotine and cotinine. By studying the action of these compounds in in vitro and in vivo systems, the role played by nicotine and related alkaloids can be better assessed and the causes and consequences of adverse effects understood.